A variety of techniques and processes have been developed to obtain genetic information, including broad genetic profiling or identifying patterns of discrete markers in genetic codes and nucleotide level sequencing of entire genomes. Knowledge of DNA sequences has become indispensible for basic biological research in numerous applied fields such as biotechnology, forensic biology, diagnostic, systematic biology, synthetic biology and personal healthcare. The advent of DNA sequencing has significantly accelerated biological research and discovery. While techniques have been developed to read, at the nucleotide level, a genetic sequence, such methods can be time-consuming and extremely costly.
Most current technologies of Next Generation Sequencing (reviewed in Metzker, M. L., NATURE REVIEWS GENETICS 11: 31-45 (2010)) have greatly lowered the cost of sequencing by shifting from electrophoresis-based methods to chip-based sequencing, which typically involves sequencing single target molecules instead of deriving sequence information from a population of amplified target molecules. The introduction of real-time sequencing, wherein the progression of successive nucleotide incorporation events is monitored while the nucleic acid polymerization process takes place, has also improved the efficiency of sequencing.
The common strategies for real-time single-molecule sequencing were derived from the concept of pyrosequencing, wherein the PPi moiety of nucleotide triphosphate monomers is labeled with a photo-detectable label. In pyrosequencing reactions, a photo-signal is released and detected during polymerase extensions as each monomer is incorporated into a growing chain (see, e.g., Ronaghi, et al., SCIENCE, 281: 363-365 (1998); Hyman, ANAL. BIOCHEM., 174: 423-436 (1988); and U.S. Pat. Nos. 6,255,083 and 7,329,492). A method of single-molecule detection using a zero-mode waveguide (ZMW) to increase the signal-to-noise ratio in single-molecule sequencing has also been described in U.S. Pat. Nos. 7,170,050 and 7,056,676.
In any enzyme-mediated, template-dependent sequencing process, the overall fidelity, processivity, and accuracy of the incorporation process can directly impact sequence determination. Lower accuracy of target sequence reads may require multiple-fold coverage to determine the sequence of a target with a high level of confidence. Despite the recent developments, a need exists for a sequencing scheme that provides greater accuracy per sequencing reaction.